The objective of this research is to investigate the critical molecular signals responsible for commitment to ionizing radiation-induced cell death. Our studies have identified a multiple-step process that is required to fully activate apoptosis. The Bcl-2 and caspase-family proteins represent the basic regulators of apoptotic cell death, with additional critical molecules identified, which assist in triggering cell death in the absence of caspases. The precise mechanism by which these proteins interact to regulate cell death in mammalian cells is unclear. Our recent studies indicate that radiation-induced apoptosis is associated with transcriptional activation of two distinct classes of pro-apoptotic bcl-2 family genes which may activate distinct steps required to fully execute the apoptotic process. In addition, irradiation leads to activation of caspase 6 and additional factors that may participate in caspase-independent cell death. To examine their unique roles in apoptosis, we will use a genetic approach to disrupt apoptosis regulatory networks and component genes by RNA interference, and use biochemical methods to determine the mechanism of activation of distinct apoptotic targets in the isogenic cell lines created. Our specific aims are: 1) To determine the mechanism of transcriptional regulation of proapoptotic Bcl-2 family genes by identifying candidates using cDNA array hybridization, examining regulation of gene expression in tumor cells and cell lines with known p53 status, and determining the mechanism of transcriptional activation by p53 using chromatin immunoprecipitation assays; 2) To determine the role of multi-domain and BH3-only proapoptotic Bcl-2-family proteins in regulating mitochondrial events, by examining their sub-cellular localization, interactions between them and with other known or novel proteins in cells in which their expression is ablated by RNA interference (RNAi); and 3) To determine the role of cysteine and serine proteases, by examining their expression and regulation, with a focus on caspase 6 and the serine protease Omi/HatrA2, in cells in which the apoptotic components have been inactivated through RNAi or expression of dominant-negative regulators. These experiments will determine the relative contribution of caspase dependent and independent apoptotic pathways to cell death. Our studies will increase our understanding of the mechanism by which critical cellular signaling molecules activate apoptosis and may provide novel strategies for overcoming resistance in radiotherapy by therapeutically enhancing proapoptotic regulators. [unreadable] [unreadable]